Role of amino acid position 70 in the binding affinity of p50.1 and p58.1 receptors for HLA-Cw4 molecules.
In an attempt to identify the amino acid position(s) of the HLA-C-specific p58.1/p50.1 natural killer cell receptors that determine the binding affinity for their ligand, we used soluble fusion proteins formed by the ectodomain of either receptor and the Fc portion of human IgG1. We show that the soluble p50.1 (activating) receptor binds weakly to 221-Cw4 transfectants. In contrast, the soluble p58.1 (inhibitory) receptor binds with high affinity. A single amino acid mutation at position 70, obtained by site-directed mutagenesis, was found to affect the binding affinity of both the p50.1 and the p58.1 receptors. Thus, substitution in p50.1 of lysine 70 by threonine (typical of the inhibitory p58.1 molecule) resulted in a dramatic increase in binding affinity, comparable to that of the p58.1 molecule. On the other hand, substitution of threonine 70 by lysine in p58.1 almost abolished binding to 221-Cw4 cells. Our present data indicate that a single amino acid difference greatly influences the p58.1/p50.1 affinity for their HLA-C ligand and suggests a possible role of position 70 as a contact site in the natural killer cell receptor/major histocompatibility complex class I interaction.
Biassoni
R
R
Istituto Nazionale per la Ricerca sul Cancro and Centro Biotecnologie Avanzate, Genova, Italy.
Pessino
A
A
Malaspina
A
A
Cantoni
C
C
Bottino
C
C
Sivori
S
S
Moretta
L
L
Moretta
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D015236:HLA-C Antigens / Q000276:immunology*; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D008969:Molecular Sequence Data; D011971:Receptors, Immunologic / Q000276:immunology*; D054340:Receptors, KIR; D054344:Receptors, KIR2DL3; D055607:Receptors, Natural Killer Cell; D016415:Sequence Alignment
D015236:HLA-C Antigens; C039966:HLA-C*04 antigen; C515078:KIR2DL3 protein, human; D011971:Receptors, Immunologic; D054340:Receptors, KIR; D054344:Receptors, KIR2DL3; D055607:Receptors, Natural Killer Cell